Our long-term objective for this proposed research is to contribute to the more comprehensive understanding of the regulation of hemoglobin synthesis and erythropoiesis under stress conditions and in red cell disorders. In this proposal, we focus on translational control by the heme-regulated eIF2? kinase (HRI) in oxidative stress and stress erythropoiesis. Our laboratory has demonstrated that HRI is necessary to reduce ineffective erythropoiesis and to maintain proper gene expression in erythroid precursors during iron deficiency. HRI is also essential for reducing the phenotypic severities of ?-thalassemia. Phosphorylation of eIF2? by HRI not only inhibits translation globally, but also selectively increases the translation of ATF4 mRNA in primary erythroid precursors. This HRI-activated ATF4 stress response pathway is necessary to mitigate oxidative stress and to promote erythroid differentiation. Most recently, HRI-eIF2?P pathway has been shown to induce fetal hemoglobin (HbF) production in human CD34+ cells undergoing erythroid differentiation. While transcriptional regulation during erythropoiesis has been studied extensively, much less is known about the role of translational control in this process. We hypothesize that eIF2 ?P-mediated translation is necessary to mitigate oxidative stress and to promote erythroid differentiation during stress erythropoiesis. We will employ a recently developed genome-wide approach, ribosome profiling, to study translational regulation during normal and stress erythropoiesis in heme deficiency and in ?-thalassemia. A novel line of erythroid-specific eIF2?Ser51Ala knockin (erythroid- ?A/A) mice, which are defective in eIF2?P signaling specifically in the erythroid lineage, will be generated. Erythroblasts isolated by FACS sorting from fetal livers of wild type (Wt), Hri-/- and erythroid-A/A mice under iron sufficient and deficint conditions, will be used to study the role of heme, HRI and eIF2?P in regulating in vivo translation genome-wide. In addition, ribosome profiling will also be performed in splenic basophilic erythroblasts of ?-thalassemic mice. The outcomes of these proposed studies will elucidate the essential role of heme and eIF2?P-mediated translation in erythropoiesis under stress conditions. This proposed research will also uncover novel molecular mechanisms in translational regulation and new proteins produced in the erythroid lineage during differentiation. The novel information obtained from these studies will advance the field of erythropoiesis greatly and will have a very significant impact on the development of new therapies for hemoglobinopathies.